(a) Field of the Invention
The invention relates to genomic probes for the detection of the presence of Candida albicans in a biological sample.
(b) Description of Prior Art
Systemic infection by fungi is an increasing problem, especially in immunocompromised and leukemic patients. Candida species are the most common of these fungal pathogen representing approximately 8% of all microorganisms recovered from blood (C. albicans account for more than 60% of these isolates). Mortality rate in these individuals with candidemia has been reported to vary between 38 to 90% depending on the underlying therapeutic procedures and immune status of the patient. A disturbingly large number of these patients die with undiagnosed invasive Candida infections.
The diagnosis of systemic candidiasis has been problematic even though patients may have extensive invasive Candida infections of several deep organs. In fact, it is evaluated that only 30 to 50% of all patients with disseminated C. albicans infections are blood culture positive (Edwards, J. E., Invasive Candida infections N. Med., 1991, 324(15):1060-1062).
Current techniques for the detection and identification of fungi mainly involve culture. This long-available method often proves too slow, cumbersome and insensitive (&gt;10.sup.3 yeast per ml) in clinical settings. Detection of a variety of antibodies and antigens in invasive candidiasis has also been extensively investigated, but with limited success. Thus, there is crucial need for a fast, accurate and effective test to diagnose deep-seated Candida infections.
U.S. Pat. No. 4,874,695, which issued on Oct. 17, 1989 to Pincus, discloses a method for identification of fungal microorganisms, characterized as "rapid". It involves culturing the microorganisms for 2 to 3 days, preparing an inoculum from the culture, mixing the inoculum with a chromogenic substrate (or separately with more than one such substrate) for detecting the presence or absence of one or more of acetate esterase, leucylglycine aminopeptidase and glycylglycine aminopeptidase by formation of a colored product or a product convertible to a colored product, and incubating the inoculum/substrate mixture(s) for 2 to 6 hours, whereby the unknown microorganism is identified by comparing with the enzyme activity of known genera and species. Thus, the overall procedure takes 2 to 3 days plus 2 to 6 hours. However, the sensitivity of this method is restricted by culture.
Other known methods are the so-called gold standard diagnosis which employs culturing in different media; and a method using monoclonal antibodies combined with latex agglutination. In a well-established method, potassium hydroxide solution is applied to a smear sample on a glass slide, whereby all cells are destroyed except Candida hyphae and spores, a microscopic examination being carried out to identify the organism. This (KOH) method is, however, subjective and requires considerable laboratory skills.
An initial step in DNA-based methods for typing Candida species and strains, is direct examination of the fluorescence pattern obtained after restriction digests of total genomic DNA and electrophoresis. These DNA fingerprints distinguish Candida species and many C. albicans subtypes (Scherer, S. et al., J. Clin. Microbiol., 1986, 2.5:675-679). One drawback of this approach is that only a limited number of sites in the genome of a given species can be scored for differences, primarily the highly conserved ribosomal DNA and the mitochondrial DNA.
Species-specific DNA probes that recognizes repetitive sequences of the Candida albicans genome were also described by Scherer et al. (P.N.A.S. USA, 1988, 85:1452-1456) and Soll et al. (J. Clin. Microbiol., 1990, 28:1236-1243). These DNA segments present the advantage of being species-specific, hybridizing with C. albicans as well as C. stellatoidea. However numerous drawbacks can be identified: the nature of these fragments remain unknown and, the patterns obtained are complex and only relatively constant in time. Moreover, these segments cannot be used as PCR targets since their complete sequences remain unknown.
Since Polymerase Chain Reaction (PCR) analysis of clinical samples is gradually becoming incorporated into diagnostic laboratory practice, this method could provide an interesting alternative to present diagnostic methods for fungi. In fact, PCR amplification of specific regions in the genome of a variety of lower eukaryotes has been shown to allow rapid identification of these microorganisms.
Recently, the polymerase chain reaction was used to detect, in clinical specimens, a DNA sequence coding for cytochrome P.sub.450 L.sub.I A.sub.I, a single copy fungus-specific gene. This Polymerase Chain Reaction (PCR) method was able to detect the presence of as few as 100 organisms per ml in a variety of clinical specimens. However, this method is not specific to any particular fungi.
Genes coding for ribosomal RNA are attractive targets for PCR based detection schemes as sensitivity is likely to be higher than in schemes designed to detect single copy genes since rDNA genes are present in multiple copies in each organism. Accordingly, a PCR method based on the detection of a segment of the 18S rRNA gene was shown to detect as few as 15 cells per ml. This target sequence was however conserved throughout the fungal kingdom.
There is still no reliable single step technique available for routine detection and species identification of the most common pathogenic genus in fungi, Candida sp. Thus, it would be highly desirable to obtain a PCR test for the inter-species discrimination of fungi.
C. albicans is a primary concern since it represents the most common fungal human pathogen in vaginal, systemic and nosocomial infections. However, identification of Candida species remains important since the incidence of non-C. albicans infections is rising, and these yeasts are not eradicated as effectively with current drug therapies.
Thus, it would be highly desirable to be provided with means to detect trace amount of Candida albicans present in a biological sample and with a suitable gene for the detection, identification and speciation of Candida sp.